Abstract Objective(s): Although several chemical and physical methods for gene delivery have been introduced, their cytotoxicity, non-specific immune responses and the lack of biodegradability remain the main issues. In this study, hydroxyapatite nanoparticles (NPs) and 1,2-dioleoyl-sn-glycero-3-phosphoethanol​amine (DOPE)-modified hydroxyapatite NPs was coated with antisense oligonucleotide of E6 mRNA, and their uptakes into the cervical cancer cell line were evaluated. Materials and Methods: Calcium nitrate and diammonium phosphate were used for the synthesis of the hydroxyapatite nanoparticle. Thus, they were coated with polyethylene glycol (PEG), DOPE and antisense oligonucleotide of E6 mRNA using a cross-linker. Then, hydroxyapatite NPs and DOPE-modified hydroxyapatite NPs were incubated 48 hours with cervical cancer cells and their uptakes were evaluated by fluorescent microscopy. Results: The hydroxyapatite NPs had different shapes and some agglomeration with average size of 100 nm. The results showed DOPE-modified hydroxyapatite NPs had higher uptake than hydroxyapatite NPs (P<0.05). Conclusions: Hydroxyapatite NPs conjugated with DOPE are a good choice for gene delivery and silencing of viral genes in cervical cancer cells, but their efficacy should be addressed more in future studies.

1.  Please cite this paper as:
Saffarzadeh N, Kalantar M, Jebali A, Hekmati moghaddam H, Sheikhha MH, Ghasemi N, Farashahi E. The cellular
uptake of antisense oligonucleotid of E6 mRNA into cervical cancer cells by DOPE-modified hydroxyapatite
nanoparticles, Nanomed J, 2015; 1(5): 292-297.
Original Research
Online ISSN 2322-5904
http://nmj.mums.ac.ir
Received: Feb. 15, 2014; Accepted: Mar. 12, 2014
Vol. 1, No. 5, Autumn 2014, page 292-297
Received: Apr. 22, 2014; Accepted: Jul. 12, 2014
Vol. 1, No. 5, Autumn 2014, page 298-301
The cellular uptake of antisense oligonucleotid of E6 mRNA into cervical
cancer cells by DOPE-modified hydroxyapatite nanoparticles
Negin Saffarzadeh1
, Seyed Mehdi Kalantar1,2*
, Ali Jebali¹*
, Seyed Hossein Hekmati
moghaddam3
, Mohammad Hassan Sheikhha1,2
, Nasrin Ghasemi1,2
, Ehsan Farashahi1,2
1
Department of Medical Genetic, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
2
Research and Clinical Centre for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
²Department of Laboratory Sciences, Faculty of Paramedicine, Shahid Sadoughi University of Medical
Sciences,Yazd, Iran
Abstract
Objective(s): Although several chemical and physical methods for gene delivery have been
introduced, their cytotoxicity, non-specific immune responses and the lack of
biodegradability remain the main issues. In this study, hydroxyapatite nanoparticles (NPs)
and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)-modified hydroxyapatite NPs
was coated with antisense oligonucleotide of E6 mRNA, and their uptakes into the cervical
cancer cell line were evaluated.
Materials and Methods: Calcium nitrate and diammonium phosphate were used for the
synthesis of the hydroxyapatite nanoparticle. Thus, they were coated with polyethylene
glycol (PEG), DOPE and antisense oligonucleotide of E6 mRNA using a cross-linker. Then,
hydroxyapatite NPs and DOPE-modified hydroxyapatite NPs were incubated 48 hours with
cervical cancer cells and their uptakes were evaluated by fluorescent microscopy.
Results: The hydroxyapatite NPs had different shapes and some agglomeration with average
size of 100 nm. The results showed DOPE-modified hydroxyapatite NPs had higher uptake
than hydroxyapatite NPs (P<0.05).
Conclusions: Hydroxyapatite NPs conjugated with DOPE are a good choice for gene
delivery and silencing of viral genes in cervical cancer cells, but their efficacy should be
addressed more in future studies.
Keyword(s): Cervical cancer cells, DOPE, Hydroxyapatite nanoparticles, Oligonucleotide
*Corresponding author: Seyed Mehdi Kalantar, Department of Medical Genetic, Shahid Sadoughi University
of Medical Sciences, Yazd, Iran. Tel: +989131518918, Email: kalantarsm@ystp.ac.ir
Ali Jebali, Department of Medical Genetic, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Tel:+989390348478, Email: alijebal2011@gmail.com

2. Oligonucleotide uptake by modified hydroxyapatite nanoparticles
Nanomed J, Vol. 1, No. 5, Autumn 2014 293
Original Research (font 12)Introduction
Nanoparticles (NPs) are solid colloidal
materials, ranging from 10 to 1000 nm
(1). NPs have sites in which the
biologically active substances are
entrapped, encapsulated, or adsorbed
onto their surface. On the other hand,
NPs were described as nanopellets and
nanocapsules, adjuvants, drug delivery
systems, and etc (2). Cationic polymer
including polybutyl-cyanoacrylate
(PBCA), poly-isohexyl-cyanoacrylate
(PIHCA), and poly-hexylcyanoacrylate
(PHCA) were first introduced as gene
delivery systems for plasmid DNA by
Bertling et al. in 1991, who compared
their efficiency with other delivery
systems (3). Based on previous studies,
oligodeoxynucleotides (ODNs) can be
bound to NPs (4-6).
Cationic polymer can be adsorbed on the
inorganic material such as
hydroxyapatite which is chemically
similar to the mineral component of
bones and hard tissues in mammals (7,
8). It is one of few materials that are
classified as bioactive, meaning that it
would support bone growth and
osseointegration when used in
orthopaedic, dental and maxillofacial
applications (9). The chemical nature of
hydroxyapatite lends itself to
substitution, meaning that it is not
uncommon for non-stoichiometric
hydroxyapatites to exist. Advanced
implants, e.g. hip replacements, dental
implants and bone conduction implants,
are coated with hydroxyapatite which
may promote osseointegration (9).
Porous hydroxyapatite can be used for
local drug delivery and for repair early
lesions in tooth enamel (10). On the other
hand, hydroxyapatite can be employed
for gene delivery same as other gene
carrier (11). To improve characteristics
of this material, some polymeric
substance may be added such as
hexadecyltrimethylammonium bromide
(CTAB) and 1,2-dioleoyl-sn-glycero-3-
phosphoethanolamine (DOPE)
(12). These are hydrophobic cationic
detergents that were used in combination
with hydrophobic polymer particles and
other NPs as adsorption enhancers for
hydrophilic anionic oligonucleotides
(13). In this study, we synthesized
hydroxy-apatite NPs coated with PEG,
modified by DOPE and covered by
antisense oligonucleotide of E6 mRNA.
Then, their uptake into cervical cancer
cells was investigated.
Materials and Methods
Materials
Calcium nitrate, ammonium phosphate,
sodium hydroxide, carbonic acid, Giemsa
and methanol were purchased from
Merck (Germany). The carboxylated
polyethylene glycol (PEG), 1-Ethyl-3-[3-
dimethy-laminopropyl] carbodiimide
hydrochloride (EDC), DOPE, and RPMI
1640 medium were obtained from
Sigma-Aldrich, USA. Antisense
oligonucleotid labeled with HEX was
purchased from Takapo Zyst Co.
Synthesis of hydroxyapatite NPs and
their modification
Calcium nitrate (36.15 g) was dissolved
in deionized water (525 mL). Di-
ammonium phosphate (12 g) was
dissolved in deionized water 375 ,L).
Then, a portion of each solution (25 mL)
was added and its pH was adjusted to pH
= 11. The mixture was incubated 6 h at
room temperature for growth process.
After incubation, NPs were washed three
times with deionized water and
centrifuged at 5000 rpm for 5 min. Then,
carboxy-PEG (1 mL) was added to
washed NPs (1 g) and incubated 30 min
at 37 °C. After incubation, NPs coated
with carboxy-PEG were centr-ifuged.
Then, 1 mL of DOPE and 1 mL of EDC
was added to NPs coated with carboxy-
PEG (1 g), incubated for 30 min at 37 °C
and centrifuged at 5000 rpm for 5 min. In
the next step, antisense oligonucleotid (1
mL, 1000 nM) of E6 mRNA was added
to DOPE-coated NPs and incubated for 1

3. Saffarzadeh N, et al
294 Nanomed J, Vol. 1, No. 5, Autumn 2014
h at 37 °C and washed by deionized
water and centrifuged at 5000 rpm for 5
min. For control, washed hydroxyapatite
NPs were directly exposed to antisense
oligonucle-otid (1 mL, 1000 nM) of E6
mRNA and incubated for 1 h at 37 °C,
washed, and centrifuged at 5000 rpm for
5 min.
Characterization of NPs
For characterization, three methods were
used including Scanning Electron
Microscope (SEM) for morphology,
Dynamic Light Scattering (DLS) for size
distribution, and Fourier transform
infrared spectroscopy (FTIR) for surface
characteristics.
Uptake of NP
100 µL of serial concentrations (500,
250, 125, 62 and 31 µg/ml) of
hydroxyapatite NPs and final modified
hydroxyapatite NPs were separately
added to 100 µL of cervical cancer cells
(103
cells) which suspended in the
RPMI1640 with 10% fetal calf serum,
and incubated for 48 hours at 37 °C.
Then, the cells were washed twice with
normal saline. Then, 100 µL of carbonic
acid (1 mL, carbonic acid) was added
and the cells were incubated for 24 h.
After incubation, the optical absorbance
(OA) of each well was read by FTIR at
2999 cm-1
and the level f uptake was
measured separately for the cells exposed
to hydroxyapatite NPs and modified
hydroxyapatite NPs. Finally, the
fluorescent of cells which incubated with
labeled hydroxyapatite NPs and modified
hydroxyapatite NPs was studied. Thus,
cells were fixed with methanol and
examined by fluorescent microscope
with magnification 1000.
Results
Characterization of NPs
The SEM image of modified
hydroxyapatite NPs is shown in the
Figure 1. As seen, they exhibited
different forms and a high degree of
agglomeration was seen. The size
distribution of modified hydroxyapatite
NPs is shown in the Figure 2. The mean,
the standard deviation and the
polydispersity index (PDI) were 100 nm,
38 nm and 0.15, respectively. The FTIR
spectrum (Figure 3) confirmed the
attachment of DOPE and oligonucleotide
to the hydroxyapatite NPs.
Figure 1. The SEM image of modified
hydroxyapatite NPs.
Figure 2. The size distribution of modified
hydroxyapatite NPs.
Uptake of NP
Figure 4 shows the uptake of
oligonucleotide by both hydroxyapatite
NPs and modified hydroxyapatite NPs.
As shown, modified hydroxyapatite NPs
had higher uptake than hydroxyapatite
NPs. In both, the pattern of uptake was
dose dependent. The microscopic
pictures of cancer cells which treated
with hydroxyapatite NPs and modified

4. Oligonucleotide uptake by modified hydroxyapatite nanoparticles
Nanomed J, Vol. 1, No. 5, Autumn 2014 295
Original Research (font 12)
Figure 3. The FTIR spectrum of oligonucleotide
(a), DOPE (b), PEG (c), hydroxyapatite NPs (d),
hydroxyapatite NPs covered by oligonucleotide (e),
modified hydroxyapatite NPs covered by
oligonucleotide (f).
hydroxyapatite NPs are shown in the
Figure 5a and Figure 5b, respectively.
Remarkably, the cancer cells which treated
with hydroxyapatite NPs had less
florescent than cells treated with modified
hydroxyapatite NPs.
Discussion
One of the challenges of the in vivo
application of gene therapies is the
efficient delivery, especially across the
plasma membrane to the cytoplasm of
target cells (14).
Figure 4. The uptake of oligonucleotide by
hydroxyapatite NPs and modified hydroxyapatite
NPs. * p<0.05 compared with modified
hydroxyapatite NPs.
Figure 5. The microscopic pictures of cancer cells
which treated with hydroxyapatite NPs (a) and
modified hydroxyapatite NPs (b).
Nanomedicine is a newly emerging
practice that fuses nanotechnology and
medicine. NPs have been used as
diagnostic probes in targeted therapy
(15). Hydroxyapatite is the principal
inorganic constituent of human bones
and teeth and its molecular formula is
Ca10(PO4)6(OH)2. Synthetic HAP crystals
are now widely used in medical implants
and as coatings on prostheses (7-9).
Compared to viral vectors which possess
risks of pathogenicity and
immunogenicity, synthetic HAP NPs
have favorable biocompatibility and high
osteoconductivity and/or osteoinductivity
without immunogenicity or pro-infla-
mmatory features (16).
HAP not only can directly inhibit the
proliferation of cancer cells but also it
can be used as a gene delivery system
due to its safety, economy and efficiency
(11, 17).
Moreover, hydroxyapatite can be
modified in order to increase its gene
delivery capability (18, 19). DOPE is a
phospholipid molecule which has a
positive charge and can be employed for
inducing positive charge on the NPs (20).
On the other hand, DOPE helps
oligonucleotide to escape from lysosome
(21). The endolysosomal escape of gene
carriers is crucial in enhancing the
efficacy of their macromolecular
payload, esp-ecially the payloads that are
susceptible to lysosomal degradation
(22). DOPE that enable the endo-
lysosomal escape of macromolecules
Wavenumber (cm-1
)
Absorbance(a.u.)

5. Saffarzadeh N, et al
296 Nanomed J, Vol. 1, No. 5, Autumn 2014
such as DNA are inte-rested by its ability
to carry various classes of genes and
therapeutic agents (23). This study
showed that the uptake of
oligonucleotide by DOPE-modified
hydroxy-apatite NPs washigher than that
of hydroxyapatite NPs alone. Both
showed a dose-dependent pattern of
uptake which is common in gene
delivery. The microscopic pictures of
cancer cells showed that cells which
treated with DOPE-modified
hydroxyapatite NPs had higher florescent
intensity than hydroxyapatite NPs alone.
Conclusions
Hydroxyapatite NPs conjugated with
DOPE are a good choice for gene
delivery aimed at silencing of genes in
cervical cancer cells and could be a
suitable carrier for the purposes of gene
therapy. Their efficacy should be
addressed more in future studies.
Acknowledgments
The authors wish to thank infertility
center and Yazd University of Medical
Sciences for their financial support.
References
1. Jiang W, Kim BY, Rutka JT, Chan WC.
Nanoparticle-mediated cellular response
is size-dependent. Nat Nanotechnol.
2008; 3(3): 145-150.
2. Mohanraj V, Chen Y. Nanoparticles - a
review. Trop J Pharm Res. 2007; 5(1):
561-573.
3. Bertling WM, Gareis M, Paspaleeva V,
Zimmer A, Kreuter J, Nurnberg E, et al.
Use of liposomes, viral capsids, and NPs
as DNA carriers. Biotechnol Appl
Biochem. 1991; 13(3): 390-405.
4. Han G, Ghosh P, Rotello VM. Multi-
functional gold nanoparticles for drug
delivery. In: Chan WCW, editor. Bio-
applications of nanoparticles. USA:
Springer; 2007. p. 48-56.
5. Tseng YC, Mozumdar S, Huang L.
Lipid-based systemic delivery of siRNA.
Adv Drug Deliv Rev. 2009; 61(9): 721-
731.
6. Yin RX, Yang DZ, Wu JZ. Nanoparticle
drug- and gene-eluting stents for the
prevention and treatment of coronary
restenosis. Theranostics. 2014; 4(2):
175-200.
7. Rezwan K, Chen QZ, Blaker JJ,
Boccaccini AR. Biodegradable and
bioactive porous polymer/inorganic
composite scaffolds for bone tissue
engineering. Biomaterials. 2006; 27(18):
3413-3431.
8. Kong L, Gao Y, Lu G, Gong Y, Zhao N,
Zhang X. A study on the bioactivity of
chitosan/nano-hydroxyapatite composite
scaffolds for bone tissue engineering.
Eur Polym J. 2006; 42(12): 3171-3179.
9. Zhou H, Lee J. Nanoscale
hydroxyapatite particles for bone tissue
engineering. Acta Biomaterialia. 2011;
7(7): 2769-2781.
10. Kim HW, Knowles JC, Kim HE.
Hydroxyapatite/poly(epsilon-
caprolactone) composite coatings on
hydroxyapatite porous bone scaffold for
drug delivery. Biomaterials. 2004; 25(7):
1279-1287.
11. Uskoković V, Uskoković DP. Nanosized
hydroxyapatite and other calcium
phosphates: chemistry of formation and
application as drug and gene delivery
agents. J Biomed Mate Res B Appl
Biomater. 2011; 96(1): 152-191.
12. Shimizu K, Ito A, Honda H. Mag-
seeding of rat bone marrow stromal cells
into porous hydroxyapatite scaffolds for
bone tissue engineering. J Biosci
Bioeng. 2007; 104(3): 171-177.
13. De Oliveira MC, Rosilio V, Lesieur P,
Bourgaux C, Couvreur P, Ollivon M, et
al. pH-sensitive liposomes as a carrier
for oligonucleotides: a physico-chemical
study of the interaction between DOPE
and a 15-mer oligonucleotide in excess
water. Biophys Chem. 2000; 87(2): 127-
137.
14. Verma IM, Somia N. Gene therapy-
promises, problems and prospects.
Nature. 1997; 389(6648): 239-242.
15. Lammers T, Hennink W, Storm G.
Tumour-targeted nanomedicines:
principles and practice. Br J Cancer.
2008; 99(3): 392-397.
16. Wang H, Li Y, Zuo Y, Li J, Ma S,
Cheng L. Biocompatibility and
osteogenesis of biomimetic nano-
hydroxyapatite/polyamide composite
scaffolds for bone tissue engineering.
Biomaterials. 2007; 28(22): 3338-3348.
17. Olton D, Li J, Wilson ME, Rogers T,
Close J, Huang L, et al. Nanostructured
calcium phosphates (NanoCaPs) for
non-viral gene delivery: influence of the
synthesis parameters on transfection

6. Oligonucleotide uptake by modified hydroxyapatite nanoparticles
Nanomed J, Vol. 1, No. 5, Autumn 2014 297
Original Research (font 12)efficiency. Biomaterials. 2007; 28(6):
1267-1279.
18. Zhu SH, Huang BY, Zhou KC, Huang
SP, Liu F, Li YM, et al. Hydroxyapatite
NPs as a novel gene carrier. J Nanopart
Res. 2004; 6(2): 307-311.
19. Chen Y, Yang L, Huang S, Li Z, Zhang
L, He J, et al. Delivery system for
DNAzymes using arginine-modified
hydroxyapatite nanoparticles for
therapeutic application in a
nasopharyngeal carcinoma model. Int J
Nanomedicine. 2013; 8: 3107-18. doi:
10.2147/IJN.S48321.
20. Navarro G, Essex S, Sawant RR, Biswas
S, Nagesha D, Sridhar S, et al.
Phospholipid-modified
polyethylenimine-based
nanopreparations for siRNA–mediated
gene silencing: Implications for
transfection and the role of lipid
components. Nanomedicine. 2014;
10(2): 411-419.
21. Akita H, Kogure K, Moriguchi R,
Nakamura Y, Higashi T, Nakamura T, et
al. Nanoparticles for ex vivo siRNA
delivery to dendritic cells for cancer
vaccines: programmed endosomal
escape and dissociation. J Control
Release. 2010; 143(3): 311-317.
22. Seong K, Seo H, Ahn W, Yoo D, Cho S,
Khang G, et al. Enhanced cytosolic drug
delivery using fully biodegradable
poly(amino oxalate) particles. J Control
Release. 2011; 152(2): 257-263.
23. Lechardeur D, Verkman A, Lukacs GL.
Intracellular routing of plasmid DNA
during non-viral gene transfer. Adv
Drug Deliv Rev. 2005; 57(5): 755-767.